The host response to periodontal pathogenic bacteria involves a complex series of events that involves both local and systemic release of cytokines that initiates and controls inflammatory reactions (Page, 1991
; Kornman et al., 1997
). In the present cross-sectional study, we investigated whether several candidate biomarkers in whole saliva, shown in previous studies to be elevated in gingival crevicular fluid of periodontitis patients, were associated with a history of alveolar bone loss. We developed a novel system to quantify whole mouth alveolar bone loss. This system was quite simple to implement and yielded data that could be correlated with salivary biomarker concentration. Further studies should be performed to validate this simplified bone score system by comparing it with measures of CEJ–alveolar crest distances.
Univariate analyses confirmed the association of a number of putative risk factors with periodontal disease (e.g. gender, age, history of osteoporosis, diabetes, and smoking). In addition, several individual biomarkers were also significantly correlated with bone loss score (IL-1β, IL-6, PGE2
, osteocalcin and osteonectin). However, multivariate analysis revealed that IL-1β and osteonectin were the only two biomarkers studied that were significantly correlated with bone loss score. A recent study found a relationship between clinical measures of periodontal disease and salivary levels of IL-1β and matrix metalloproteinase (MMP)-8 (Miller et al., 2006
). The results of the present study are consistent with that report. Unfortunately, however, this present study did not quantify (MMP)-8 in saliva.
In the present study, a higher concentration of IL-1β in patient saliva was associated with higher alveolar bone loss score. In contrast, a higher concentration of osteonectin in patient saliva was associated with a lower alveolar bone loss score. While these differences were found to be statistically significant, there were a number of discrepancies noted. For example, several subjects having high salivary levels of IL-1β showed no evidence of bone loss. The negative association between bone loss and osteonectin is somewhat surprising in light of previous studies that reported this protein to be elevated in GCF of periodontitis subjects (Bowers et al., 1989
The concentrations of TNF-α and IL-6 in the saliva of subjects were mostly below the limit of detection (IL-6 was still, however, significantly correlated to alveolar bone loss score in the univariate analysis). There are several possible explanations for these findings. First, there might be competitive inhibitors in saliva that interfere with quantitation of the analyte. Second, the results may be influenced by the ability of the antibody pair to recognize and bind the specific epitopes on the target biomarkers. Because these antibodies are produced against recombinant cytokines, they may not exhibit exactly the same epitopes as natural cytokines. Third, saliva samples might contain proteases that could degrade the cytokines and decreased their detection.
The biomarker concentrations measured in this study are somewhat less than those reported by other recent publications, For example, while the present study reports mean TNF-α concentrations to be 1.9±0.2 pg mL−1
in whole saliva for both men and women, Wozniak et al
. reported TNF-α levels to be much higher [median of 127.1 pg mL−1
(range 40.4–1594.9)] in whole saliva from a population that did not include anyone with any form of periodontitis and gingivitis (Wozniak et al., 2002
). In contrast, like this present study, Aurer and coworkers found that TNF was below the level of detection in almost all samples (Aurer et al., 2005
). Similarly, the IL-1β concentrations in whole saliva of 123.5±144.5 pg mL−1
reported in this study are considerably lower than those reported by Miller et al
. (212.8±167.4 pg mL−1
in control subjects and 753.7±1022.4 pg mL−1
in those with moderate-severe periodontitis) (Miller et al., 2006
). There are several potential explanations for these discrepancies. First, the studies used different methods to collect saliva. Wozniak et al
. collected unstimulated whole saliva that was centrifuged for 5 min at 800 g
and filter sterilized. Miller et al
. collected unstimulated saliva, with subjects rinsing their mouths with tap water before saliva collection. Also, they did not centrifuge the saliva. In the present study, saliva was centrifuged for a longer period of time [and under greater force than the above-mentioned studies (6000 g
for 10 min)]. Both actions would reduce concentrations of the target biomarkers in saliva. Obviously, there is a need for standardization of saliva collection so that the results from different studies can be compared. The important point is that the relative relationships within each study be considered. In this case, there are similar trends between studies (for example with respect to the elevation of IL-1β in subjects with alveolar bone loss).
Involvement of IL-1β in the pathogenesis of periodontal disease has long been suspected because of similarities between the known biological effects of the cytokine and the manifestations of the disease (Tatakis, 1993
). Although both isoforms of IL-1, IL-1α and IL-1β have similar biological activities, IL-1β is more potent in stimulating bone resorption and is the form more frequently found in GCF (Stashenko et al., 1987
; Masada et al., 1990
). The specific role of osteonectin in periodontal pathogenesis has not yet been determined. However, it has been found to be the dominant noncollagenous protein of mineralized tissues and has been identified in several other tissues as well (Termine et al., 1981
; Nomura et al., 1988
From multivariate analysis, age was strongly correlated to alveolar bone loss in the present study. A previous study found that after age 50 years, the porosity of the mandibular alveolar cortical bone increases, while at the same time there is a decrease in bone mass (Hildebolt, 1997
). It has been suggested that this increase in alveolar bone porosity in combination with local factors could be of etiologic importance in the rate of periodontal alveolar bone loss.
Smoking may be one of the most significant risk factors in the development of periodontal disease (Tomar & Asma, 2000
). Our findings also indicate that subjects who smoke have higher alveolar bone loss scores. Consistent with previous studies (Palomo et al., 2005
), subjects taking bisphosphonate drugs, approved for the treatment and prevention of postmenopausal osteoporosis, showed lower alveolar bone loss scores than those not taking these drugs.
The possibility of using saliva as a diagnostic tool for the determination of health status is dependent upon several factors such as sensitivity of detection and stability of biomarker in saliva and recovery of sufficient amounts to be detectable. Preliminary studies designed to evaluate the effect of whole saliva and parotid saliva on exogenous cytokines were conducted immediately following the addition of known amount(s) of cytokine. Compared to cytokines added to PBS-TBN, the detection of cytokines in whole saliva was much reduced (c
. 75% compared to control), similar to findings previously reported by Wozniak and coworkers (Wozniak et al., 2002
). This reduction in detection is also similar to that observed for cytokines in cervical mucus (Ginsburg et al., 1997
), which, like saliva, contains substantial amounts of mucins that may sequester biomarkers to limit their detection. We have made no effort to adjust the clinical values we measured for each biomarker in saliva ().
Inhibitors of serine and cysteine proteases, as well as of aspartic and aminopeptidases, did not reverse the observed reduction in cytokines following addition to saliva. Thus, it is possible that the reduction in cytokine detection may be due to complexation of the molecules with salivary components such as mucins (Iontcheva et al., 1997
A previous study to assess the stability of cytokines in whole saliva samples showed that addition of cytokines (IL-12, IFN-γ, IL-4 and IL-10) to whole saliva with incubation for 48 h at −80°, 4°, 21° or 37 °C showed no significant difference in biomarkers levels compared to samples tested at time 0 (Wozniak et al., 2002
). Different incubation times and multiple freeze-thaw cycles might change the physiological state of whole saliva, for example viscosity, and thus might affect the detection of cytokines. However, our results suggested that incubation time and multiple freeze-thaw cycles did not affect biomarker detection.
Quantitation of biomarkers in saliva may serve as a useful tool to monitor and predict susceptibility to periodontal disease. The practical significance of this cross-sectional study remains to be determined. The sequestering and/or degradation of possible biomarkers in saliva may limit the detection of the absolute concentration of each biomarker. In the case of cytokines targeted in our study, the true concentration of biomarkers in saliva may be greater than that detected by the immunoassays used. Future, longitudinal well-controlled clinical trials are needed to find reliable salivary biomarkers for periodontal diagnosis and to determine future risk of disease.